Method of Loading and Distributing Cells in a Bioreactor of a Cell Expansion System

ABSTRACT

One or more embodiments are described directed to a method and system for loading and distributing cells in a bioreactor of a cell expansion system. Accordingly, embodiments include methods and systems that may provide for adding a plurality of cells to a fluid within a bioreactor of the cell expansion system. A first percentage of the plurality of cells is allowed to settle in the bioreactor and a second percentage of the plurality of cells is allowed to settle outside of the bioreactor. The first percentage of cells is then expanded in the bioreactor. The second percentage of cells is wasted.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 61/691,193, entitled METHOD OF LOADING AND DISTRIBUTINGCELLS IN A BIOREACTOR OF A CELL EXPANSION SYSTEM, filed on Aug. 20,2012, and hereby incorporated by references in its entirety as if setforth herein in full.

BACKGROUND

CESs are used to expand and differentiate cells. Cell expansion systemsare known in the art. For example, U.S. Pat. Nos. 5,162,225 and6,001,585 generally describe cell expansion systems designed for cellexpansion.

The potential use of stem cells in a variety of treatments and therapieshas achieved particular attention. Cell expansion systems can be used toexpand, e.g., grow, stem cells, as well as other types of cells, such asbone marrow cells. Stem cells which are expanded from donor cells can beused to repair or replace damaged or defective tissues and have broadclinical applications for a wide range of diseases. Recent advances inthe regenerative medicine field demonstrates that stem cells haveproperties such as proliferation and self-renewal capacity, maintenanceof the unspecialized state, and the ability to differentiate intospecialized cells under particular conditions.

Cell expansion systems include one or more compartments for expandingthe cells, such as a cell growth chamber, also referred to herein as a“bioreactor.” In order to expand cells, an initial volume of cells istypically loaded into, and distributed within, the bioreactor.Accordingly, there is a need for a method of loading and distributingcells in a bioreactor associated with a cell expansion system. Thepresent disclosure addresses this and other needs.

Embodiments of the present invention have been made in light of theseand other considerations. However, the relatively specific problemsdiscussed above do not limit the applicability of the embodiments of thepresent invention to solving other problems.

SUMMARY

The summary is provided to introduce aspects of some embodiments of thepresent invention in a simplified form, and is not intended to identifykey or essential elements of the claimed invention, nor is it intendedto limit the scope of the claims.

It is to be understood that the present invention may include a varietyof different versions or embodiments, and this Summary is not meant tobe limiting or all-inclusive. This Summary provides some generaldescriptions of features that may be included in embodiments, and alsoinclude some more specific descriptions of other features that may beincluded in other embodiments.

One or more embodiments are generally directed to a method and systemfor loading and distributing cells in a bioreactor of a cell expansionsystem. Accordingly, embodiments include methods that may provide foradding a plurality of cells to a fluid circulating at a first ratewithin a bioreactor of the cell expansion system. The circulation rateof the fluid is maintained at a first rate for a predetermined period oftime to obtain a desired cell concentration in the fluid. After thedesired cell concentration has been achieved, the circulation rate ofthe fluid is reduced to a reduced rate that is less than the first rate.A first percentage of the plurality of cells is allowed to settle in thebioreactor and a second percentage of the plurality of cells is allowedto settle outside of the bioreactor. The first percentage of cells isthen expanded in the bioreactor. The second percentage of cells iswasted. While the circulation rate is maintained at the first rate,embodiments may provide for rotating the bioreactor around a rotationalaxis from a first orientation to a second orientation, pausing thebioreactor at the second orientation for a first period of time,rotating the bioreactor back around the rotational axis to the firstorientation, and then pausing the bioreactor back at the firstorientation for a second period of time.

Embodiments of the present invention are also directed to a system forexpanding cells. The embodiments may include a bioreactor reactorcomprising a first fluid flow path having at least opposing ends, afirst opposing end of said first fluid flow path fluidly associated witha first port of a hollow fiber membrane and a second end of said firstfluid flow path fluidly associated with a second port of the hollowfiber membrane, wherein said first fluid flow path comprises anintracapillary portion of said hollow fiber membrane. The embodimentsmay also include a fluid inlet path fluidly associated with said firstfluid flow path, wherein the plurality of cells are introduced into thefirst fluid flow path through the first fluid inlet path. A first pumpfor circulating fluid in the first fluid flow path of the bioreactor mayalso be included in the embodiments. The embodiments may further includea controller for controlling operation of the first pump, wherein thecontroller is configured to control the pump to circulate a fluid at afirst rate within the first fluid flow path, maintain the circulationrate of the fluid at the first rate for a predetermined period of timeto obtain a desired cell concentration in the fluid, after the desiredcell concentration has been achieved, reduce the circulation rate of thefluid to a reduced rate that is less than the first rate to allow afirst percentage of the plurality of cells to settle in the bioreactorand a second percentage of the plurality of cells to settle outside ofthe bioreactor, and circulate a fluid with growth media for expandingthe first percentage of cells in the bioreactor and removing most of thesecond percentage of cells from the first fluid flow path.

As used herein, “at least one,” “one or more,” and “and/or” areopen-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “oneor more of A, B, or C” and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

Various embodiments of the present inventions are set forth in theattached figures and in the Detailed Description as provided herein andas embodied by the claims. It should be understood, however, that thisSummary does not contain all of the aspects and embodiments of the oneor more present inventions, is not meant to be limiting or restrictivein any manner, and that the invention(s) as disclosed herein is/are andis understood by those of ordinary skill in the art to encompass obviousimprovements and modifications thereto.

Additional advantages of the embodiments presented herein will becomereadily apparent from the following discussion, particularly when takentogether with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1A depicts one embodiment of a cell expansion system (CES);

FIG. 1B depicts another embodiment of a CES;

FIG. 1C depicts yet another embodiment of a CES;

FIG. 1D depicts a rocking device for moving a cell growth chamberrotationally or laterally during operation of the CES;

FIG. 1E depicts a detachable flow circuit for use with a CES;

FIG. 2A depicts a side view of a hollow fiber cell growth chamberembodiment of a cell growth chamber;

FIG. 2B depicts a cut-away side view of the hollow fiber cell growthchamber embodiment of FIG. 2A;

FIG. 3 is a front elevation view of an embodiment of a bioreactorshowing circulation paths through the bioreactor;

FIG. 4 is a perspective view of a portion of a cell expansion system,including a detachably attached bioreactor;

FIG. 5 is a flow chart of a method associated with loading anddistributing cells in a cell expansion system;

FIG. 6 is a front elevation view a bioreactor in a first orientation;

FIG. 7 is a front elevation view of the bioreactor of FIG. 6, whereinthe bioreactor is shown rotated through 90 o of rotation;

FIG. 8 is a front elevation view of the bioreactor of FIG. 6, whereinthe bioreactor is shown rotated through 180 o of rotation;

FIG. 9 is a front elevation view of the bioreactor of FIG. 6, whereinthe bioreactor is shown rotated through 270 o of rotation and to asecond position;

FIG. 10 is a front elevation view of the bioreactor of FIG. 6, whereinthe bioreactor is shown rotated back to the initial starting position;

FIG. 11 is a perspective view of a bioreactor connected to a shaftassembly of a CES;

FIG. 12 is a side elevation view of the structures shown in FIG. 11;

FIG. 13 is a detail perspective view of a fitting used to rotate abioreactor around its longitudinal axis;

FIG. 14 is a side elevation view of a bioreactor illustrating rotationin roll;

FIG. 15 is a graph showing expanded cells harvested after being loadedand distributed using previous methods;

FIG. 16 is a graph showing expanded cells harvested after being loadedand distributed using methods consistent with embodiments of the presentinvention;

FIG. 17 is a graph showing lactate generation during expansion for cellsthat have been loaded and distributed using methods consistent withembodiments of the present invention;

FIG. 18 is a graph showing glucose consumption during expansion forcells that have been loaded and distributed using methods consistentwith embodiments of the present invention;

FIG. 19 is a graph showing lactate generation during expansion for cellsafter being loaded and distributed using previous methods;

FIG. 20 is a graph showing average lactate generation during expansionafter being loaded and distributed using previous methods;

FIG. 21 illustrates a block diagram of a basic computer that may be usedto implement embodiments of the present invention; and

FIG. 22 is a flow chart of a method associated with loading anddistributing cells in a cell expansion system according to anembodiment;

DETAILED DESCRIPTION

The principles of the present invention may be further understood byreference to the following detailed description and the embodimentsdepicted in the accompanying drawings. It should be understood thatalthough specific features are shown and described below with respect todetailed embodiments, the present invention is not limited to theembodiments described below. The present disclosure is generallydirected to a method for distributing a plurality of cells in abioreactor of a cell expansion system. As described below, a method ofdistributing cells within a bioreactor may include loading cells intothe bioreactor, rotating the bioreactor, and holding the bioreactorstill at certain orientations.

A schematic of an example cell expansion system (CES) is depicted inFIG. 1A. CES 10 includes first fluid circulation path 12 and secondfluid circulation path 14. First fluid flow path 16 has at leastopposing ends 18 and 20 fluidly associated with a hollow fiber cellgrowth chamber 24 (also referred to herein as a “bioreactor”).Specifically, opposing end 18 is fluidly associated with a first inlet22 of cell growth chamber 24, and opposing end 20 is fluidly associatedwith first outlet 28 of cell growth chamber 24. Fluid in firstcirculation path 12 flows through the interior of hollow fibers ofhollow fiber membrane 50 disposed in cell growth chamber 24 (cell growthchambers and hollow fiber membranes are described in more detail infra).Further, first fluid flow controller 30 is operably connected to firstfluid flow path 16, and controls the flow of fluid in first circulationpath 12.

Second fluid circulation path 14 includes second fluid flow path 34,cell growth chamber 24, and a second fluid flow controller 32. Thesecond fluid flow path 34 has at least opposing ends 36 and 38. Opposingends 36 and 38 of second fluid flow path 34 are fluidly associated withinlet port 40 and outlet port 42 respectively of cell growth chamber 24.Fluid flowing through cell growth chamber 24 is in contact with theoutside of hollow fiber membrane in the cell growth chamber 24. Secondfluid circulation path 14 is operably connected to second fluid flowcontroller 32.

First and second fluid circulation paths 12 and 14 are thus separated incell growth chamber 24 by a hollow fiber membrane. Fluid in first fluidcirculation path 12 flows through the intracapillary (“IC”) space of thehollow fibers in the cell growth chamber. First circulation path 12 isthus referred to as the “IC loop.” Fluid in second circulation path 14flows through the extracapillary (“EC”) space in the cell growthchamber. Second fluid circulation path 14 is thus referred to as the “ECloop.” Fluid in first fluid circulation path 12 can flow in either aco-current or counter-current direction with respect to flow of fluid insecond fluid circulation path 14.

Fluid inlet path 44 is fluidly associated with first fluid circulationpath 12. Fluid inlet path 44 allows fluid into first fluid circulationpath 12, while fluid outlet path 46 allows fluid to leave CES 10. Thirdfluid flow controller 48 is operably associated with fluid inlet path44. Alternatively, third fluid flow controller 48 can alternatively beassociated with fluid outlet path 46.

Fluid flow controllers as used herein can be a pump, valve, clamp, orcombination thereof. Multiple pumps, valves, and clamps can be arrangedin any combination. In various embodiments, the fluid flow controller isor includes a peristaltic pump. In further embodiments, fluidcirculation paths, inlet ports, and outlet ports can be constructed oftubing of any material.

Various components are referred to herein as “operably associated.” Asused herein, “operably associated” refers to components that are linkedtogether in operable fashion, and encompasses embodiments in whichcomponents are linked directly, as well as embodiments in whichadditional components are placed between the two linked components.“Operably associated” components can be “fluidly associated.” “Fluidlyassociated” refers to components that are linked together such thatfluid can be transported between them. “Fluidly associated” encompassesembodiments in which additional components are disposed between the twofluidly associated components, as well as components that are directlyconnected. Fluidly associated components can include components that donot contact fluid, but contact other components to manipulate the system(e.g. a peristaltic pump that pumps fluids through flexible tubing bycompressing the exterior of the tube).

Generally, any kind of fluid, including buffers, protein containingfluid, and cell-containing fluid can flow through the variouscirculations paths, inlet paths, and outlet paths. As used herein,“fluid,” “media,” and “fluid media” are used interchangeably.

FIG. 1B depicts a more detailed cell expansion system 800. CES 800includes a first fluid circulation path 802 (also referred to as the“intracapillary loop” or “IC loop”) and second fluid circulation path804 (also referred to as the “extracapillary loop” or “EC loop”). Firstfluid flow path 806 is fluidly associated with cell growth chamber 801to form first fluid circulation path 802. Fluid flows into cell growthchamber 801 through IC inlet port 801A, through hollow fibers in cellgrowth chamber 801, and exits via IC outlet port 801B. Pressure sensor810 measures the pressure of media leaving cell growth chamber 801. Inaddition to pressure, sensor 810 may in embodiments also be atemperature sensor that detects the media pressure and temperatureduring operation. Media flows through IC circulation pump 812 which canbe used to control the rate of media flow. IC circulation pump 812 maypump the fluid in a first direction or second direction opposite thefirst direction. Exit port 801B can be used as an inlet in the reversedirection. Media entering the IC loop may enter through valve 814. Asthose skilled in the art will appreciate, additional valves and/or otherdevices can be placed at various locations to isolate and/or measurecharacteristics of the media along portions of the fluid paths.Accordingly, it is to be understood that the schematic shown representsone possible configuration for various elements of the CES 800 andmodifications to the schematic shown are within the scope of the one ormore present embodiments.

With regard to the IC loop, samples of media can be obtained from samplecoil 818 during operation. Media then returns to IC inlet port 801A tocomplete fluid circulation path 802. Cells grown/expanded in cell growthchamber 801 can be flushed out of cell growth chamber 801 into harvestbag 899 through valve 898 and line 897. Alternatively, when valve 898 isclosed, the cells may be redistributed within chamber 801 for furthergrowth.

Fluid in second fluid circulation path 804 enters cell growth chamber801 via EC inlet port 801C, and leaves cell growth chamber 801 via ECoutlet port 801D. Media in the EC loop is in contact with the outside ofthe hollow fibers in the cell growth chamber 801, thereby allowingdiffusion of small molecules into and out of the hollow fibers that maybe within chamber 801.

Pressure/temperature sensor 824 disposed in the second fluid circulationpath 804 allows the pressure and temperature of media to be measuredbefore the media enters the EC space of the cell growth chamber 801.Sensor 826 allows the pressure and temperature of media in the secondfluid circulation path 804 to be measured after it leaves the cellgrowth chamber 801. With regard to the EC loop, samples of media can beobtained from sample port 830 or a sample coil during operation.

After leaving EC outlet port 801D of cell growth chamber 801, fluid insecond fluid circulation path 804 passes through EC circulation pump 828to gas transfer module 832. EC circulation pump 828 may also pump thefluid in opposing directions. Second fluid flow path 822 is fluidlyassociated with gas transfer module 832 via an inlet port 832A and anoutlet port 832B of gas transfer module 832. In operation, fluid mediaflows into gas transfer module 832 via inlet port 832A, and exits gastransfer module 832 via outlet port 832B. Gas transfer module 832 addsoxygen to and removes bubbles from media in the CES 800. In variousembodiments, media in second fluid circulation path 804 is inequilibrium with gas entering gas transfer module 832. The gas transfermodule 832 can be any appropriately sized device known in the art anduseful for oxygenation or gas transfer. Air or gas flows into gastransfer module 832 via filter 840 and out of oxygenator or gas transferdevice 832 through filter 838. Filters 838 and 840 reduce or preventcontamination of oxygenator 832 and associated media. Air or gas purgedfrom the CES 800 during portions of a priming sequence can vent to theatmosphere via the gas transfer module 832.

In the configuration depicted for CES 800, fluid media in first fluidcirculation path 802 and second fluid circulation path 804 flows throughcell growth chamber 801 in the same direction (a co-currentconfiguration). The CES 800 can also be configured to flow in acounter-current conformation.

In accordance with at least one embodiment, media, including cells (froma source such as a cell container, e.g. a bag) can be attached atattachment point 862, and fluid media from a media source can beattached at attachment point 846. The cells and media can be introducedinto first fluid circulation path 802 via first fluid flow path 806.Attachment point 862 is fluidly associated with the first fluid flowpath 806 via valve 864, and attachment point 846 is fluidly associatedwith the first fluid flow path 806 via valve 850. A reagent source maybe fluidly connected to point 844 and be associated with fluid inletpath 842 via valve 848, or second fluid inlet path 874 via valves 848and 872.

Air removal chamber (ARC) 856 is fluidly associated with firstcirculation path 802. The air removal chamber 856 may include one ormore sensors including an upper sensor and lower sensor to detect air, alack of fluid, and/or a gas/fluid interface, e.g., an air/fluidinterface, at certain measuring positions within the air removal chamber856. For example, ultrasonic sensors may be used near the bottom and/ornear the top of the air removal chamber 856 to detect air, fluid, and/oran air/fluid interface at these locations. Embodiments provide for theuse of numerous other types of sensors without departing from the spiritand scope of the present disclosure. For example, optical sensors may beused in accordance with embodiments of the present disclosure. Air orgas purged from the CES 800 during portions of the priming sequence orother protocols can vent to the atmosphere out air valve 860 via line858 that is fluidly associated with air removal chamber 856.

An EC media source may be attached to EC media attachment point 868 anda wash solution source may be attached to wash solution attachment point866, to add EC media and/or wash solution to either the first or secondfluid flow path. Attachment point 866 may be fluidly associated withvalve 870 that is fluidly associated with first fluid circulation path802 via valve 872 and first fluid inlet path 842. Alternatively,attachment point 866 can be fluidly associated with second fluidcirculation path 804 via second fluid inlet path 874 and second fluidflow path 884 by opening valve 870 and closing valve 872. Likewise,attachment point 868 is fluidly associated with valve 876 that may befluidly associated with first fluid circulation path 802 via first fluidinlet path 842 and valve 872. Alternatively, fluid container 868 may befluidly associated with second fluid inlet path 874 by opening valve 876and closing valve distribution 872.

In the IC loop, fluid may be initially advanced by the IC inlet pump854. In the EC loop, fluid is initially advanced by the EC inlet pump878. An air detector 880, such as an ultrasonic sensor, may also beassociated with the EC inlet path 884.

In at least one embodiment, first and second fluid circulation paths 802and 804 are connected to waste line 888. When valve 890 is opened, ICmedia can flow through waste line 888 and to waste bag 886. Likewise,when valve 892 is opened, EC media can flow to waste bag 886.

After cells have been grown in cell growth chamber 801, they may beharvested via cell harvest path 897. Here, cells from cell growthchamber 801 can be harvested by pumping the IC media containing thecells through cell harvest path 897, with valve 898 open, into cellharvest bag 899.

Various components of the CES 800 can be contained or housed within amachine or housing, such as a cell expansion machine, wherein themachine maintains cells and media at a predetermined temperature. It isfurther noted that in embodiments, components of CES 800 may be combinedwith other CES such as CES 10 (FIG. 1A) or CES 900 (FIG. 1C). In otherembodiments, a CES may include fewer components than shown in FIGS. 1A-Cand still be within the scope of the present disclosure.

FIG. 1C depicts another embodiment of a CES. CES 900 includes firstfluid circulation path 902 (also referred to as the “intracapillary (IC)loop”) and second fluid circulation path 904 (also referred to as the“extracapillary loop” or “EC loop”).

First fluid flow path 906 is fluidly associated with cell growth chamber908 to form first fluid circulation path 902. Fluid flows into cellgrowth chamber 908 through inlet port 910, through hollow fibers in cellgrowth chamber 908, and exits via outlet port 907. Pressure gauge 917measures the pressure of media leaving cell growth chamber 908. Mediaflows through valve 913 and pump 911, which can be used to control therate of media flow. Samples of media can be obtained from sample port905 or sample coil 909 during operation. Pressure/temperature gauge 915disposed in first fluid circulation path allows detection of mediapressure and temperature during operation. Media then returns to inletport 910 to complete fluid circulation path 902. Cells expanded in cellgrowth chamber 908 can be flushed out of cell growth chamber 908 orredistributed within hollow fibers for further growth.

Second fluid circulation path 904 includes second fluid flow path 912that is fluidly associated with cell growth chamber 908 in a loop. Fluidin second fluid circulation path 904 enters cell growth chamber 908 viainlet port 914, and leaves cell growth chamber 908 via outlet port 916.Media is in contact with the outside of the hollow fibers in the cellgrowth chamber 908, allowing diffusion of small molecules into and outof the hollow fibers.

Pressure/temperature gauge 919 disposed in the second circulation pathallows the pressure and temperature of media to be measured before themedia enters the EC space of the cell growth chamber 908. Pressure gauge921 allows the pressure of media in the second circulation path to bemeasured after it leaves the cell growth chamber.

After leaving outlet port 916 of cell growth chamber 908, fluid insecond fluid circulation path 904 passes through pump 920 and valve 922to oxygenator 918. Second fluid flow path 912 is fluidly associated withoxygenator 918 via oxygenator inlet port 924 and oxygenator outlet port926. In operation, fluid media flows into oxygenator 918 via oxygenatorinlet port 924, and exits oxygenator 918 via oxygenator outlet port 926.

Oxygenator 918 adds oxygen to media in the CES. In various embodiments,media in second fluid circulation path 904 is in equilibrium with gasentering oxygenator. The oxygenator can be any oxygenator known in theart. Gas flows into oxygenator 918 via filter 928 and out of oxygenator918 through filter 930. Filters 928 and 930 reduce or preventcontamination of oxygenator 918 and associated media.

In the configuration depicted for CES 900, fluid media in firstcirculation path 902 and second circulation path 904 flow through cellgrowth chamber 908 in the same direction (a co-current configuration).Those of skill in the art will recognize that CES 900 can also beconfigured in a counter-current conformation. Those of skill in the artwill recognize that the respective inlet and outlet ports can bedisposed in the cell growth chamber at any location.

Cells and fluid media can be introduced to fluid circulation path 902via first fluid inlet path 932. Fluid container 934 and fluid container936 are fluidly associated with first fluid inlet path 932 via valves938 and 940 respectively. Likewise, cell container 942 is fluidlyassociated with first fluid circulation path 902 via valve 943. Cellsand fluid proceed through heat exchanger 944, pump 946, and into dripchamber 948. Drip chamber 948 is fluidly associated with firstcirculation path 902. Overflow from drip chamber 948 can flow out ofdrip chamber 948 from overflow line 950 via valve 952.

Additional fluid can be added to first or second fluid circulation paths902 and 904 from fluid container 954 and fluid container 956. Fluidcontainer 954 is fluidly associated with valve 958 which is fluidlyassociated with first fluid circulation path 902 via first fluid inletpath 960. First fluid flow path includes valve 964. Alternatively, fluidcontainer 954 is fluidly associated with second fluid inlet path 962.Likewise, fluid container 956 is fluidly associated with valve 966,which is fluidly associated with first fluid circulation path 902 viafirst fluid inlet path 960. Alternatively, fluid container 956 isfluidly associated with second fluid inlet path 962.

Second fluid inlet path 962 is configured to allow fluid to flow throughpump 968 before entering drip chamber 970. Second fluid inlet path 962continues to second fluid circulation path 904. Overflow fluid can flowout via overflow line 972 through valve 974 to waste container 976.

Cells can be harvested via cell harvest path 978. Cells from cell growthchamber 908 can be harvested by pumping media containing the cellsthrough cell harvest path 978 to cell harvest bag 980.

First and second fluid circulation paths 902 and 904 are connected byconnector path 984. When valve 986 is opened, media can flow throughconnector path 984 between first and second circulation paths 902 and904. Likewise, pump 990 can pump media through another connector path988 between first and second fluid circulation paths 902 and 904.

Various components of the CES can be contained within incubator 999.Incubator 999 maintains cells and media at a constant temperature.

As will be recognized by those of skill in the art, any number of fluidcontainers (e.g. media bags) can be fluidly associated with the CES inany combination. It will further be noted that the location of the dripchamber, or sensors independent of the drip chamber, can be at anylocation in the CES before inlet port 910.

The CES can include additional components. For example, one or more pumploops (not shown) can be added at the location of peristaltic pumps onthe CES. The pump loops may be made of polyurethane (PU) (available asTygothane C-210A)). Alternatively, a cassette for organizing the tubinglines and which may also contain tubing loops for the peristaltic pumpsmay also be included as part of the disposable.

A detachable flow circuit (also referred to herein as a “detachablecirculation module”) may also be provided in some embodiments. Thedetachable flow circuit may be a portion of a cell expansion moduleconfigured to attach to a more permanent fixed portion of the CES.Generally, the fixed portions of the CES include peristaltic pumps. Invarious embodiments, the fixed portions of the CES can include valvesand/or clamps.

The detachable flow circuit can include a first fluid flow path havingat least two ends. The first end is configured to be fluidly associatedwith a first end of a cell growth chamber, and a second end of the firstfluid flow path configured to fluidly associated with a second end ofthe cell growth chamber.

Likewise, the detachable flow circuit can include a second fluid flowpath having at least two ends. Portions of the detachable flow circuitcan be configured to be fluidly associated with an oxygenator and/orbioreactor. The detachable flow circuit can include a second fluid flowpath that may be configured to fluidly associate with the oxygenator andcell growth chamber.

In various embodiments, the detachable flow circuit may be detachablyand disposably mounted to a fluid flow controller. The detachable flowcircuit can include detachable fluid conduits (e.g. flexible tubing)that connects portions of the CES. With reference to FIG. 1E, thedetachable flow circuit includes the tubing for first fluid circulationpath 126, but without a pump. The detachable flow circuit can furtherinclude the tubing for flush line 132, without a valve. The detachableflow circuit can further include the tubing connecting first circulationpath 126 to flush line 132, and first fluid inlet path 124. In variousother permutations, the detachable flow circuit can include tubing thatconnects the media inlet bags 106 and 108, vent bag 110, and cell inputbag 112 to drip chamber 186. The detachable flow circuit can alsoinclude tubing connecting cell harvest bag 140 to first circulation path126.

Likewise, the detachable flow circuit can include tubing that makes upsecond circulation path 166. For example, the tubing can include tubingconnecting oxygenator 104 to cell growth chamber 102, as well as dripchamber 186. The detachable flow circuit can also include fluid inletpath 114.

In further embodiments, the detachable flow circuit can include a cellgrowth chamber, oxygenator, as well as bags for containing media andcells. In various embodiments, the components can be connected together,or separate. Alternatively, detachable flow circuit can include one ormore portions configured to attach to fluid flow controllers, such asvalves, pumps, and combinations thereof. In variations where peristalticpumps are used, the detachable circuit module can include a peristalticloop configured to fit around a peristaltic portion of the tubing. Invarious embodiments, the peristaltic loop can be configured to befluidly associated with the circulations paths, inlet paths, and outletpaths. The detachable flow circuit can be combined in a kit withinstructions for its assembly or attachments to fluid flow controllers,such as pumps and valves.

Embodiments provide for using a number of different methods to introducecells into bioreactors of CES. As described in greater detail below,embodiments include methods and systems that distribute cells in thebioreactor to promote consistent expansion of cells.

According to embodiments, cells can be grown (“expanded”) in either theIC loop or the EC loop. Adherent and non-adherent suspension cells canbe expanded. In one embodiment, the lumen of the cell growth chamberfibers can be coated with fibronectin. Divalent cation-free (e.g.calcium and magnesium-free) PBS is added to a CES system. After adherentcells are introduced into a cell growth chamber, e.g., chamber 24, 908,or 801 they are incubated for a sufficient time to adhere to the hollowfibers. IC and EC media are circulated to ensure sufficient nutrientsare supplied to the cells.

The flow rate of the IC loop and EC loop can be adjusted to a specificvalue. In various embodiments, the flow rate of the IC loop and EC loopscan be, independently set to, about 2, about 4, about 6, about 8, about10, about 15, about 20, about 25, about 30, about 35, about 40, about45, about 50, about 60, about 70, about 80, about 90, about 100, about200, about 300, about 400 or about 500 mL/minute. In variousembodiments, the flow rates for the IC circuit loop may be 10-20mL/minute, and the flow rate of the EC circuit loop may be 20-30 mL perminute (allowing media to flow through an oxygenator and re-establishoxygen levels). Additional media may be pumped into the CES at a lowerflow rate (e.g. 0.1 mL per minute in some embodiments) to replace mediathat evaporates through a gas exchange module(s) such as gasexchange/oxygenators 30, 918, or 832. In various embodiments, the ECloop removes cellular waste, and the IC loop includes growth factors inthe media.

CES's may provide a great deal of flexibility in varying growthconditions and criteria. Cells can be kept in suspension in the IC loopby circulating media continuously. Alternatively, media circulation canbe stopped, causing cells to settle. Fresh media can be added to the ICloop by ultrafiltration to accommodate excess volume without removingcells. EC media circulation allows for exchange of gas, nutrients, wasteproducts, and addition of new media without removing cells.

Expanded cells can include adherent cells, non-adherent cells, or aco-culture of any combination of cells in the art.

In embodiments, to harvest adherent cells, the IC and EC media may bereplaced with media that is free of divalent cations (e.g. divalentcation-free PBS). In one embodiment, trypsin may be loaded into a firstcirculation path, and allowed to incubate with adherent cells for aperiod of time (in some embodiments about 5 to about 10 minutes). Thetrypsin may then be flushed from the system. A shearing force may beapplied to the cells by increasing the flow rate through cell growthchamber, and adherent cells that are released from the cell growthchamber may be pumped to a cell harvest bag.

When non-adherent cells are expanded, the cells can be flushed from thecirculating IC circuit. Adherent cells remain in the cell growthchamber, while non-adherent cells are removed.

The CES can be used to perform a variety of cell expansion methods.

In one embodiment, a seeded population of cells can be expanded. Cellsare introduced, or seeded, into the CES. In certain circumstances, thelumen of the hollow fibers can be conditioned to allow cell adhesion.Cells are then added to the cell growth chamber, and adherent cellsadhere to the hollow fibers, while non-adherent cells (e.g. hematopoeticstem cells, or HSCs) do not adhere. The non-adherent cells can beflushed from the system. After incubation for a period of time, theadherent cells can be released and harvested.

Stem cells, progenitor cells, and fully differentiated cells can all beexpanded.

The cell growth chamber of the cell expansion system in embodimentsinclude a hollow fiber membrane comprised of a plurality ofsemi-permeable hollow fibers separating first and second fluidcirculation paths.

An embodiment of a cell growth chamber is depicted in FIGS. 2B and 2A,which depicts a cut-away and side view of a hollow fiber cell growthchamber 200. Cell growth chamber 200 is bounded by cell growth chamberhousing 202. Cell growth chamber housing 202 further includes fouropenings, or ports: inlet port 204, outlet port 206, inlet port 208, andoutlet port 210.

Fluid in the first circulation path enters cell growth chamber 200through inlet port 204, passes into and through the intracapillary sideof a plurality of hollow fibers (referred to in various embodiments asthe intracapillary (“IC”) side or “IC space” of a hollow fibermembrane), and out of cell growth chamber 200 through outlet port 206.The terms “hollow fiber,” “hollow fiber capillary,” and “capillary” areused interchangeably. A plurality of hollow fibers are collectivelyreferred to as a “membrane.” Fluid in the second circulation path flowsin the cell growth chamber through inlet port 208, comes in contact withthe outside of the hollow fibers (referred to as the “EC side” or “ECspace” of the membrane), and exits cell growth chamber 200 via outletport 210. Cells can be contained within the first circulation path orsecond circulation path, and can be on either the IC side or EC side ofthe membrane.

Although cell growth chamber housing 202 is depicted as cylindrical inshape, it can have any other shape known in the art. Cell growth chamberhousing 202 can be made of any type of biocompatible polymeric material.Various other cell growth chamber housings may differ in shape and size.

Those of skill in the art will recognize that the term cell growthchamber does not imply that all cells being grown or expanded in a CESare grown in the cell growth chamber. In many embodiments, adherentcells can adhere to membranes disposed in the growth chamber, or maygrow within the associated tubing. Non-adherent cells (also referred toas “suspension cells”) can also be grown. Cells can be grown in otherareas within the first or second fluid circulation path.

For example, the ends of hollow fibers 212 can be potted to the sides ofthe cell growth chamber by a connective material (also referred toherein as “potting” or “potting material”). The potting can be anysuitable material for binding the hollow fibers 212, provided that theflow of media and cells into the hollow fibers is not obstructed andthat liquid flowing into the cell growth chamber through the IC inletport flows only into the hollow fibers. Exemplary potting materialsinclude, but are not limited to, polyurethane or other suitable bindingor adhesive components. In various embodiments, the hollow fibers andpotting may be cut through perpendicular to the central axis of thehollow fibers at each end to permit fluid flow into and out of the ICside. End caps 214 and 216 are disposed at the end of the cell growthchamber.

Fluid entering cell growth chamber 200 via inlet port 208 is in contactwith the outside of hollow fibers. This portion of the hollow fiber cellgrowth chamber is referred to as the “extracapillary (EC) space.” Smallmolecules (e.g. water, oxygen, lactate, etc.) can diffuse through thehollow fibers from the interior of the hollow fiber to the EC space, orfrom the EC space to the IC space. Large molecular weight molecules suchas growth factors are typically too large to pass through the hollowfibers, and remain in the IC space of the hollow fibers. In embodimentsin which cells are grown in the IC space, the EC space is used as amedium reservoir to supply nutrients to the cells and remove thebyproducts of cellular metabolism. The media may be replaced as needed.Media may also be circulated through an oxygenator to exchange gasses asneeded.

In various embodiments, cells can be loaded into the hollow fibers byany of a variety of methods, including by syringe. The cells may also beintroduced into the cell growth chamber from a fluid container, such asa bag, which may be fluidly associated with the cell growth chamber.

Hollow fibers are configured to allow cells to grow in theintracapillary space (i.e. inside the hollow fiber lumen) of the fibers.Hollow fibers are large enough to allow cell adhesion in the lumenwithout substantially impeding the flow of media through the hollowfiber lumen. In various embodiments, the inner diameter of the hollowfiber can be greater than or equal to about 10000, about 9000, about8000, about 7000, about 6000, about 5000, about 4000, about 3000, about2000, about 1000, about 900, about 800, about 700, about 650, about 600,about 550, about 500, about 450, about 400, about 350, about 300, about250, about 200, about 150, or about 100 microns. Likewise, the outerdiameter of the hollow fiber can be less than or equal to about 10000,about 9000, about 8000, about 7000, about 6000, about 5000, about 4000,about 3000, about 2000, about 1000, about 900, about 800, about 700,about 650, about 700, about 650, about 600, about 550, about 500, about450, about 400, about 350, about 300, about 250, about 200, about 150,or about 100 microns. The hollow fiber wall thickness should besufficient to allow diffusion of small molecules, in some embodiments.

Any number of hollow fibers can be used in a cell growth chamber,provided the hollow fibers can be fluidly associated with the inlet andoutlet ports of the cell growth chamber. In various embodiments, thecell growth chamber can include a number of hollow fibers greater thanor equal to about 1000, about 2000, about 3000, about 4000, about 5000,about 6000, about 7000, about 8000, about 9000, about 10000, about 11000or about 12000. In other embodiments, the cell growth chamber caninclude a number of hollow fibers less than or equal to about 12000,about 11000, about 10000, about 9000, about 8000, about 7000, about6000, about 5000, about 4000, about 3000, or about 2000. In othervarious embodiments, the length of the hollow fibers can be greater thanor equal to about 100, about 200, about 300, about 400, about 500, about600, about 700, about 800, or about 900 millimeters. In embodiments, thecell growth chamber contains approximately about 9000 hollow fibers thathave an average length of about 295 mm, an average inner diameter of 215microns, and an average outer diameter of 315 microns.

Hollow fibers can be constructed of any material capable of forming asize sufficient to form fibers capable of transporting liquid from thecell growth chamber inlet port to the cell growth chamber outlet port.In various embodiments, the hollow fibers can be constructed fromplastic adherent materials capable of binding to certain types of cells,such as adherent stem cells (e.g. MSCs). In various other embodiments,hollow fibers can be treated with compounds such as fibronectin to formadherent surfaces.

In certain embodiments, the hollow fibers may be made of asemi-permeable, biocompatible polymeric material. One such polymericmaterial which can be used is a blend of polyamide, polyarylethersulfoneand polyvinylpyrrolidone (referred to herein as “PA/PAES/PVP”). Thesemi-permeable membrane allows transfer of nutrients, waste anddissolved gases through the membrane between the EC space and IC space.In various embodiments, the molecular transfer characteristics of thehollow fiber membranes are chosen to minimize loss of expensive reagentsnecessary for cell growth such as growth factors, cytokines etc. fromthe hollow fiber, while allowing metabolic waste products to diffusethrough the membrane into the hollow fiber lumen side to be removed.

In certain variations, one outer layer of each PA/PAES/PVP hollow fibermay be characterized by a homogenous and open pore structure with adefined surface roughness. The openings of the pores may be in the sizerange of about 0.5 to about 3 microns, and the number of pores on theouter surface of the fibers may be in the range of about 10,000 to about150,000 pores per mm2. This outer layer has a thickness of about 1 toabout 10 microns. The next layer in each hollow fiber may be a secondlayer having the form of a sponge structure and, in embodiments have athickness of about 1 to about 15 microns. This second layer may serve asa support for the outer layer. A third layer next to the second layermay have the form of finger-like structures. This third layer providesmechanical stability and a high void volume which gives the membrane alow resistance to transporting molecules through the membrane. Duringuse, the finger-like voids are filled with fluid and the fluid gives alower resistance for diffusion and convection than a matrix with asponge-filled structure having a lower void volume. This third layer mayhave a thickness of about 20 to about 60 microns.

In further embodiments, the hollow fiber membrane can include betweenabout 65 to about 95% by weight of at least one hydrophobic polymer andbetween about 5 to about 35% by weight of at least one hydrophilicpolymer. The hydrophobic polymer may be chosen from the group consistingof polyamide (PA), polyaramide (PAA), polyarylethersulphone (PAES),polyethersulphone (PES), polysulphone (PSU), polyarylsulphone (PASU),polycarbonate (PC), polyether, polyurethane (PUR), polyetherimide andcopolymer mixtures of any of the above polymers, such aspolyethersulphone or a mix of polyarylethersulphone and polyamide. Inadditional embodiments, the hydrophilic polymer may be chosen from thegroup consisting of polyvinylpyrrolidone (PVP), polyethylene glycol(PEG), polyglycolmonoester, water soluble cellulosic derivates,polysorbate and polyethylene-polypropylene oxide copolymers.

Depending upon the type of cells to be expanded in the cell growthchamber, the polymeric fibers may be treated with a substance, such asfibronectin, to enhance cell growth and/or adherence of the cells to themembrane.

The CES can include a device configured to move or “rock” the cellgrowth chamber relative to other components of the cell expansion systemby attaching it to a rotational and/or lateral rocking device. FIG. 1Dshows one such device, in which a bioreactor 400 is rotationallyconnected to two rotational rocking components, and a lateral rockingcomponent.

A first rotational rocking device component 402 rotates the bioreactoraround central axis 410 of the bioreactor and laterally connects tolateral rocking device 404. Rotational rocking device component 402 isrotationally associated to bioreactor 400. The rotational rocking devicethen rotates bioreactor 400 around central axis 410 of the bioreactor.Rotation can occur in a clockwise or counter-clockwise direction.Bioreactor 400 can be rotated continuously in a single direction aroundcentral axis 410 in a clockwise or counterclockwise direction.Alternatively, bioreactor 400 can rotate in alternating fashion, firstclockwise, then counterclockwise around central axis 410.

The CES can also include a second rotational rocking component thatrotates bioreactor 400 around rotational axis 412. Rotational axis 412passes through the center of point of bioreactor 400 and is normal tocentral axis 410. Bioreactor 400 can be rotated continuously in a singledirection around rotational axis 412 in a clockwise or counterclockwisedirection. Alternatively, bioreactor 400 can be rotated aroundrotational axis 412 in an alternating fashion, first clockwise, thencounterclockwise. In various embodiments, bioreactor 400 can also berotated around rotational axis 412 and positioned in a horizontal orvertical orientation relative to gravity.

Lateral rocking component 404 is laterally associated with bioreactor400. The plane of lateral rocking component 404 moves laterally in the−x and −y directions. The settling of cells in the bioreactor is therebyreduced movement of cell-containing media within the hollow fibers.

The rotational and/or lateral movement of the rocking device can reducethe settling of cells within the device and reduce the likelihood ofcells becoming trapped within a portion of the bioreactor. The rate ofcells settling in the cell growth chamber is proportional to the densitydifference between the cells and the suspension media according toStoke's Law. In certain embodiments, a 180 degree rotation (fast) with apause (having a total combined time of 30 seconds) repeated as describedabove keeps non-adherent red blood cells suspended. A minimum rotationof about 180 degrees is performed in some embodiments; however, onecould use rotation of up to 360 degrees or greater in other embodiments.Different rocking components can be used separately, or can be combinedin any combination. For example, a rocking component that rotatesbioreactor 400 around central axis 410 can be combined with the rockingcomponent that rotates bioreactor 400 around axis 412. Likewise,clockwise and counterclockwise rotation around different axes can beperformed independently in any combination.

With reference now to FIG. 3, an example of another cell growth chamber,bioreactor 300, is shown in front elevation view. Bioreactor 300 has alongitudinal axis LA-LA and includes bioreactor housing 304. In at leastone embodiment, bioreactor housing 304 includes four openings or ports:IC inlet port 308, IC outlet port 320, EC inlet port 328, and EC outletport 332.

Fluid in a first circulation path enters bioreactor 300 through IC inletport 308 at a first longitudinal end 312 of the bioreactor 300, passesinto and through the intracapillary side (referred to in variousembodiments as the intracapillary (“IC”) side or “IC space” of a hollowfiber membrane) of a plurality of hollow fibers 316, and out ofbioreactor 300 through IC outlet port 320 located at a secondlongitudinal end 324 of the bioreactor 300. Fluid in a secondcirculation path flows in the bioreactor 300 through EC inlet port 328,comes in contact with the extracapillary side or outside (referred to asthe “EC side” or “EC space” of the membrane) of the hollow fibers 316,and exits bioreactor 300 via EC outlet port 332. Fluid enteringbioreactor via an EC inlet port 328 is in contact with the outside ofthe hollow fibers. Small molecules (e.g. water, oxygen, lactate, etc.)can diffuse through the hollow fibers from the interior of the hollowfiber to the EC space, or from the EC space to the IC space. Largemolecular weight molecules such as growth factors are typically toolarge to pass through the hollow fibers, and remain in the IC space ofthe hollow fibers. The media may be replaced as needed. Media may alsobe circulated through an oxygenator to exchange gasses as needed. Cellscan be contained within the first circulation path and/or secondcirculation path, and can be on either the IC side and/or EC side of themembrane. By way of example and not limitation, in one embodiment, thebioreactor 300 may include about 11520 fibers that have about 215×10⁻⁶ minner diameters (ID).

Although bioreactor housing 304 is depicted as cylindrical in shape, itcould have a variety of shapes, such as a rectangular cube. Bioreactorhousing 304 can be made of any type of biocompatible polymeric material,including a substantially transparent material that permits an observerto see one or more of the plurality of hollow fibers 316, as well asfluid residing within the bioreactor housing 304. Various otherbioreactor housings may differ in shape and size.

Referring now to FIG. 4, a portion of a CES 430 is shown in perspectiveview, and includes a back portion 434 of body 408 of the CES 430. Forclarity, the front portion the body 408 is not shown; however, the frontportion is attached to the back portion 434, such as by hinges 438,thereby allowing the front portion to comprise a door or hatch that canbe opened to access the bioreactor 300 of the CES 430. Attached to thebioreactor 300 may be a spool 416 for tubing and a sampling port 420.The environment in the vicinity of the bioreactor 300 is temperaturecontrolled to provide appropriate conditions for cell growth.

Referring now to FIG. 5, a flow chart is shown that depicts oneembodiment of a cell expansion process 500 associated with using a CES,including the steps associated with loading and distributing cells inthe bioreactor 300, as further described herein. Although features ofCES 430 are described as performing some of the steps of process 500,the present invention is not limited thereto. Indeed, other CES withdifferent features, not described herein, may be utilized in someembodiments of process 500. Accordingly, reference to feature of CES 430such as bioreactor 300 are provided for illustrative purposes only, andthe process 500 is not limited to use with CES 430.

To start the cell expansion process 500, at 504 a bioreactor 300 and anyassociated tubing and related structures are attached to the body 408 toprovide an operable CES 430. Once attached to the body 408, thebioreactor 300 and its associated tubing and related structures areprimed at 508 using an appropriate priming fluid, such as saline. At512, cells are loaded and distributed in the bioreactor 300. The loadingand distributing of cells in embodiments involves a number of substeps,for example, in some embodiments step 512 additionally includesorienting the bioreactor 300 in a starting position at 516, and thenloading and distributing the cells in the bioreactor 300 at 520.Following loading and distributing cells in the bioreactor 300, thecells undergo expansion at 528. That is, the cells within the bioreactor300 are allowed to grow and/or multiply. At 532, an assessment is madeas to whether additional cells need to be added to the bioreactor 300and/or whether the bioreactor 300 needs to be rotated to distributecells within the bioreactor 300. If additional cells need to be loadedinto the bioreactor 300 and/or if cells need to be distributed in thebioreactor 300, then the cell expansion process 500 returns to step 512.If cells do not need to be added and/or the bioreactor 300 does not needto be rotated, then at 536 an assessment is made as to whether the cellexpansion process 528 is complete. As used herein, the cell expansionprocess is determined to be complete if a sufficient number of cellsand/or change in cell characteristics has been achieved. If the cellexpansion process 528 is complete, the cells are harvested at 540. Ifcell expansion process 528 is not complete, then the cell expansionprocess at 528 is allowed to continue.

With further reference to the flow chart of FIG. 5, additional detail isnow provided regarding loading and distributing cells in the bioreactor,as shown at 512. More particularly, at 516, the bioreactor 300 isoriented in its starting position. As best seen in FIG. 6, in at leastone embodiment the bioreactor 300 is positioned horizontally to initiateloading and distributing cells in the bioreactor 300. That is, after thebioreactor 300 is primed at 508, at 516 the bioreactor 300 is orientedwith its longitudinal axis LA-LA in a starting position, such as asubstantially horizontal position. Thereafter, at 520 a plurality ofcells is loaded into the bioreactor 300 while the bioreactor 300 isrotated (as described below) in a particular sequence to facilitatedistribution of the cells through the bioreactor 300.

In at least one embodiment, cells may be loaded into the IC side of thebioreactor 300 (or into the hollow fibers 316 of the bioreactor 300) bycausing flow of a media carrying the cells to pass from the IC inlet 308to the EC outlet 332. In addition, cells may be loaded into the EC sideof the bioreactor 300 (or to the exterior of the hollow fibers 316 ofthe bioreactor 300) by causing flow of a media carrying the cells topass from the EC inlet 328 to the IC outlet 320.

To assist with determining the desired movements of the bioreactor 300to facilitate improved distribution of cells within the bioreactor 300,a series of calculations may be performed, for one embodiment, tocalculate a basis for positioning the bioreactor 300. More particularly,by rotating the bioreactor 300, the influence of the acceleration due togravity on a given cell (e.g., bone marrow cell) within the bioreactor300 can be affected relative to the geometry of the bioreactor 300. Toachieve a net impulse of zero on the cell, calculation of the impulse(“I”) may be performed to determine the change during rotation andcounteract the impulse with the appropriate pause time at 0° and 270°.

To start, initial consideration may be given to the accelerationexperienced by a cell within the bioreactor 300. As a premise ofembodiments of the present invention, it may be desirable to counteractthe acceleration due to gravity (“g”) on a given cell in the bioreactor300 associated with distributing cells in the bioreactor 300.Accordingly, a rotation sequence for the bioreactor 300 is sought toachieve a net gravitational influence on a given cell of zero associatedwith loading and distributing cells in the bioreactor 300. Table 1 belowprovides a summary of gravitational acceleration influences along twoaxes, namely, the T and 11 axes as shown in FIG. 6, associated with thebioreactor 300.

TABLE 1 Summary Table Of Acceleration Directions Bioreactor Positiona_(T) a{acute over (_(L))}{acute over (_(L))} At 0° −(g is purely 0along T axis) While Rotating +(a component of −(a component of g 0° to90° g is along T axis) is along ĹĹ axis) While Rotating +(a component of−(a component of g 90° to 180° g is along T axis) is along ĹĹ axis)While Rotating +(a component of +(a component of g 180° to 270° g isalong T axis) is along ĹĹ axis) Paused at 270° 0 +(g is purely along ĹĹaxis) While Rotating +(a component of +(a component of g 270° to 180° gis along T axis) is along ĹĹ axis) While Rotating +(a component of −(acomponent of g 180° to 90° g is along T axis) is along ĹĹ axis) WhileRotating −(a component of −(a component of g 90° to 0° g is along Taxis) is along ĹĹ axis)

For Table 1, a positive sign “+” indicates acceleration in the positivedirection for the subject axis when the bioreactor is at the position orwhile rotating as shown in column 1; a negative sign “−” indicatesacceleration in the negative direction for the subject axis when thebioreactor is at the position or while rotating as shown in column 1;and zero “0” indicates substantially no acceleration for the subjectaxis when the bioreactor is at the position as shown in column 1. Zerodegrees (0°) is defined as the orientation of the bioreactor 300 whenthe longitudinal axis LA-LA is oriented horizontally with the EC inlet328 and EC outlet 332 oriented upwards (as shown in FIG. 6); 90° isdefined as vertical with the EC inlet 328 and EC outlet 332 oriented tothe left (as shown in FIG. 7); 180° is defined as the longitudinal axisLA-LA oriented horizontally with the EC inlet 328 and EC outlet 332oriented downwards (as shown in FIG. 8); and 270° is defined as verticalwith the EC inlet 328 and EC outlet 332 oriented to the right (as shownin FIG. 9).

A method of distributing cells in a bioreactor 300 includes manipulatingthe orientation of the bioreactor 300, such that a net impulse due togravity acting on cells loaded into the bioreactor 300 is substantiallyzero. In accordance with at least one embodiment, the manipulation ofthe bioreactor comprises both rotating the bioreactor 300 and thereafterholding the bioreactor stationary for set periods of time. In accordancewith at least one embodiment, the time for holding the bioreactor 300stationary t_(p) is approximately equal to the quantity 2ω⁻¹, whereinthe angular velocity ω (rad/sec) is substantially constant for theperiods when the bioreactor 300 is undergoing rotation. As those skilledin the art will appreciate, different angular velocities and pause timescan be used.

Referring again to FIG. 6, and in accordance with at least oneembodiment, the orientation of the bioreactor 300 at the initialstarting position is shown. Here, the longitudinal axis LA-LA of thebioreactor is substantially horizontal. While loading cells into thebioreactor 300, a sequence of manipulations is undertaken to mitigatethe influence of gravity on the cells loaded into the bioreactor 300.More particularly, in embodiments, the bioreactor 300 is rotated thoughapproximately 270° at a first angular velocity ω. FIG. 7 illustrates thebioreactor 300 rotated through 90°. Continuing, FIG. 8 illustrates thebioreactor 300 having been rotated through 180°. Finally, FIG. 9illustrates the bioreactor oriented at a second orientation, where thebioreactor 300 is held still for a period of time to allow the fullinfluence of gravity to act in the positive direction of the ĹĹ axis.After the appropriate period of time for pausing the bioreactor 300, thebioreactor 300 is then rotated back to its original or initial startingposition, as shown in FIG. 10.

As those skilled in the art will appreciate, more than one rotationaldirection is possible. In addition, more than one initial startingposition is also possible provided a balancing of the influence ofgravity on the cells loaded into the bioreactor 300 is achieved.Accordingly, the calculations, examples and discussion herein provideone or more possible configurations for manipulating the bioreactor 300to reduce, minimize or eliminate the influence of gravity on cells andimprove distribution of cells within the bioreactor. However, to theextent that other embodiments and variations are encompassed by thepresent disclosure, the calculations, description and figures are to beconsidered exemplary and non-limiting.

In accordance with at least one embodiment, the influence of gravity oncell distribution in the bioreactor 300 is controlled by the angularvelocity applied to the bioreactor 300. More particularly, therotational or angular velocity ω (rad/sec) is used to balance the netimpulse due to gravity experienced by cells within the bioreactor 300.

In accordance with at least one embodiment, a method of distributingcells within a bioreactor 300 having a longitudinal axis LA-LA includes:initiating the loading and distributing of cells into the bioreactor 300when the longitudinal axis LA-LA is substantially horizontal or angledat about 45° relative to horizontal; rotating the bioreactor 300 througha total of approximately 540° of angular displacement; and holding thebioreactor 300 still at a plurality of orientations. In at least oneembodiment, the angular velocity of rotation is substantially the samefor those intervals of time wherein the bioreactor is rotating. In atleast one embodiment, the angular velocity of rotation of the bioreactor300 is changed from a first angular velocity ω₁ to a second angularvelocity ω₂ for portions of the time the bioreactor 300 is undergoingrotation.

Referring now to FIGS. 11 and 12, different views of the bioreactor 300are shown with the bioreactor 300 interconnected to a shaft assembly 600by a chamber coupling 604. Motor 608 serves to rotate the outer shaft612 around a rotation axis oriented through the shaft 612 andsubstantially perpendicular to the longitudinal axis of the bioreactor300, thereby rotating the bioreactor 300 in a pitch mode as illustratedin FIGS. 7-10. Motor 616 serves to rotate an inner shaft (see FIG. 13)located within the outer shaft 612 to cause a roll fitting within thechamber coupling 604 to rotate the bioreactor 300 around itslongitudinal axis LA-LA. As best seen in FIG. 13, an inner shaft 700includes structure for engaging a roll collar 704 residing within thechamber coupling 604. The inner shaft member 700 includes a beveledpinion 708 residing at the very distal end of the inner shaft member700, and the beveled pinion 708 contacts a sloped surface 712 of theroll collar 704 such that when the inner shaft member 700 is rotated,the roll collar 704 rotates, thereby causing the cell growth chamber 300to rotate about its longitudinal axis LA-LA.

With reference now to FIG. 14, an example of rotating the cell growthchamber in the roll mode is illustrated. In FIG. 14, a side elevationview of the cell growth chamber 300 is shown, wherein in a first rollposition (shown with solid lines), the EC inlet port 328 is orientedvertically upwards. In a second roll position (shown with dashed lines),the EC inlet port 328 is oriented downwards. It is to be understood thatthe roll of the cell growth chamber 300 can be selectively controlledsuch that the cell growth chamber 300 can be rotated at any angle aroundits longitudinal axis. Periodic rotation of the cell growth chamber 300in roll assists in preventing colonies of cells from settling during thecell loading and distribution process at step 512 depicted in the flowchart shown in FIG. 5.

In at least one embodiment, cells are loaded and distributed throughoutthe bioreactor 300 during a loading and distribution step that operatesfor greater than about 2 minutes of time. In at least one embodiment theloading and distribution step may operate for several minutes. Duringthe loading and distribution step the bioreactor 300 undergoes aplurality of rotational sequences that are undertaken consecutively fromthe time loading of the cells is commenced until such time assubstantially all of the cells have been loaded into the bioreactor 300and its associated tubing.

In at least one embodiment, a bioreactor 300 is loaded with a pluralityof cells while undergoing rotation such that a net impulse due togravity acting on the plurality of cells is reduced relative to a netimpulse due to gravity acting on the plurality of cells if thebioreactor 300 was not undergoing rotation. That is, the methodcomprises manipulating an orientation of the bioreactor such that anactual net impulse due to gravity acting on the plurality cells in thebioreactor is reduced relative to an avoided net impulse due to gravityacting on the plurality cells if the bioreactor was held in a stationaryposition.

In at least one embodiment, the bioreactor 300 is rotated through atleast 180° of rotation to reduce the net impulse of gravity acting onthe plurality of cells.

In at least one embodiment, the bioreactor 300 is rotated in a pitchmode to reduce the net impulse due to gravity acting on the plurality ofcells, wherein an axis of rotation is oriented transversely to alongitudinal axis LA-LA of the bioreactor 300. In at least oneembodiment, the bioreactor 300 is rotated in a roll mode to reduce thenet impulse due to gravity acting on the plurality of cells, wherein anaxis of rotation is oriented substantially parallel to a longitudinalaxis LA-LA of the bioreactor 300. Here, the axis of rotation that issubstantially parallel to the longitudinal axis LA-LA may be coincidentwith the longitudinal axis LA-LA.

In at least one embodiment, harvesting of cells from the bioreactor 300is performed by manipulating the orientation of the bioreactor 300 asdescribed herein. That is, the bioreactor 300 is rotated to reduce a netimpulse due to gravity acting on the cells during the harvestingprocedure. Such manipulation of the bioreactor during cell harvestingimproves the collection efficiency of cells. In addition, suchmanipulation of the bioreactor during cell harvesting also improves thenumber of cells collected because the influence of gravity is overcomeas the cells are washed from the bioreactor 300.

In at least one embodiment wherein cells are grown in a suspension andnot adhered to the walls of hollow fibers in the bioreactor, thebioreactor can be continuously manipulated to reduce the influence dueto gravity on cells residing with the bioreactor.

Cells can be added to the CES by a number of methods. As noted above,with respect to FIG. 5, loading and distributing cells may involve anumber of steps or substeps. FIG. 22 illustrates a process 1100 forexpanding/growing cells that includes a number of steps for loading anddistributing cells in a cell growth chamber or bioreactor. The stepsbelow may be described with respect to features of a system, but thepresent invention is not limited thereto. In other embodiments, thesteps may be performed by other components of CES. Accordingly, thesteps of process 1100 are not limited to being performed by anyparticular structure.

Process 1100 being at 1104. At step 1108, cells are added to fluidcirculating in a cell growth chamber. In one embodiment, the cells areloaded into an intracapillary (“IC”) space loop of a bioreactor (e.g.,bioreactor 300) and uniformly suspended using circulation. Fluid in theIC loop may be circulating while the cells are added into the IC spaceloop. At step 1112 the cells are circulated though a cell growth chamberat a first rate. The circulation of the fluid may be performed using acirculation pump.

At step 1116, the circulation rate is maintained for a predeterminedperiod of time. In some embodiments, the circulation pump may becirculating the fluid in the IC loop at one rate when the cells areintroduced (e.g., step 1108), and then circulate the fluid at a secondhigher rate after the cells are introduced. In these embodiments, step1116 involves maintaining the circulation at the second higher rate.

Once cells are uniformly suspended, step 1120 is performed to reduce thecirculation rate. Step 1120 may involve reducing the rate of acirculation pump or completely stopping the pump. In any case, the rateat which the fluid is circulating is reduced at step 1120. At step 1124,the cells are then allowed to settle. As will be appreciated, the cellsare approximately uniformly distributed throughout the system, e.g., CES10, 900, 800. At step 1124, cells located within the bioreactor willsettle in the bioreactor and cells outside of the bioreactor will settleoutside of the bioreactor.

At step 1128, cells that settled in the bioreactor at step 1124, areexpanded in the bioreactor. Step 1128 may involve circulating fluid,such as media and other nutrients to the bioreactor to promote the cellgrowth. The cells that settled outside of the bioreactor will be wasted,as they will not be in the bioreactor under the conditions that promotetheir growth. Accordingly, at step 1132, the cells that settled outsideof the bioreactor at step 1124 are wasted. By the term “wasted” it ismeant that the cells are not subjected to conditions that are optimizedto promote growth of the cells, as occurs in the bioreactor. Therefore,the wasted cells are not expanded in the bioreactor, and in someembodiments the wasted cells die and/or are washed away in fluidcirculating in a CES. The wasted cells may in some embodiments expand orgrow in other parts of a CES, and in some embodiments they may even beharvested, but they will not be subjected to the same conditions,optimized for their growth, as cells in the bioreactor. The process endsat 1136.

In some embodiments process 1100 is performed in a system where thevolume in the bioreactor is from about 65% to about 70%, such as about69%, of the volume containing cells, e.g., the volume of the IC loop.When the cells are settled at step 1124, from about 30% to about 35%,such as about 31%, of the cells (located in the volume outside of the ICloop) will be wasted.

The cells that are distributed in a bioreactor, according to embodimentsof process 1100, are less influenced by the distribution of the cellssince they are generally evenly distributed in the bioreactor, and theloss of wasted cells does not result in reduced numbers of expandedcells compared to conventional methods of loading cells.

In conventional methods of loading cells in the bioreactor, steps wereperformed to attempt to push cells that settled in the volume of the ICloop, which is not within the bioreactor, into the volume within thebioreactor. The circulation of liquid in the IC loop was changed so thatcells were chased from the input line and the output line into thebioreactor by flowing fluid from the lines into both the input andoutput ends of the bioreactor. Without being bound by theory, it isbelieved that this increased the amount of cells in the bioreactor, butdid not provide an even distribution of cells. The even distribution ofcells, as provided with the method described above (process 1100),creates a more favorable environment for cell expansion and makes up forthe smaller initial amount of cells distributed in the bioreactorcompared to the previous methods.

It is noted that the method described above is provided merely forillustrative purposes. Other variations are within the scope of thepresent invention. For example, the IC loop may be such that thepercentage of cells that are deposited in the bioreactor is equal to orgreater than about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, or about 95 percent of the cellsintroduced into the IC loop of a CES. In some embodiments, the IC loopof a CES may be such that the amount of cells that are deposited in thebioreactor is less than or equal to about 95, about 90, about 85, about80, about 75, about 70, about 65, or about 60 percent of the cellsintroduced into the IC loop of a CES.

As noted above, in some embodiments the circulation rate of thecirculation pump can change during various steps of process 1100. Forexample, the circulation rate may be at a first rate when the cells areintroduced into the IC loop, step 1108. The rate may then be increasedafter the cells are introduced into the IC loop. In other embodiments,the circulation pump may be off, or at a low circulation rate, when thecells are introduced into the IC loop and then turned on, or increased,after the cells are introduced. In embodiments, the circulation rates inthe IC loop, during various steps of the methods, may be greater than orequal to about 25 ml/min, about 50 ml/min, about 75 ml/min, about 100ml/min, about 125 ml/min, about 130 ml/min, about 135 ml/min, about 140ml/min, about 145 ml/min, or about 150 ml/min. In embodiments, thecirculation rates in the IC loop, during various steps of the methods,may be less than or equal to about 400 ml/min, about 375 ml/min, about350 ml/min, about 325 ml/min, about 300 ml/min, 275 ml/min, about 250ml/min, about 225 ml/min, about 200 ml/min, about 175 ml/min, about 170ml/min, about 165 ml/min, about 160 ml/min, about 155 ml/min, 150ml/min, 145 ml/min, or 140 ml/min.

In some embodiments, in addition to circulation of the IC loop to evenlydistribute the cells in the IC loop volume, the bioreactor may also berotated to further evenly suspend the cells before the circulation pumpis turned off, or is set at a low circulation rate. The rotation of thebioreactor may be performed as described above with respect to FIGS.3-14, before, during, and/or after cells are introduced into the IC loopvolume.

EXAMPLES

In this example, hMSC cells are expanded in a bioreactor using a loadingand distribution method similar to the methods described above.

Day 0-5 Expansion of the cells: 13.6M to 177M; CV=10%; dT=32.7 hrs

Day 5-6 Expansion: 177M to 296M; CV=10%; dT=32.4 hrs

Day 6-7 Expansion: 296M to 408M; CV=1%; dT=51.7 hrs

Cells in flask culture at approximately 80% confluence at Day 7, harvest20,160 cells/cm2. In a CES, 19,429 cells/cm2 are harvested at Day 7.This may be an indication of approximately 80% confluence in thebioreactor.

The approximately 80% confluent indicator, the elongation of the celldoubling time from Day 6 to Day 7 (32 hrs to 51 hrs), and decrease in CV% all suggest the cells are being slowed by cell-cell interactions andthe capacity of the bioreactor is approaching the maximum for thisparticular cell population.

FIG. 15 illustrates cell harvest numbers as a function of lactategeneration rate for cells that are loaded and distributed usingconventional methods. FIG. 16 illustrates cell harvest numbers as afunction of lactate generation rate for cells that are loaded anddistributed using a method consistent with the methods described above,i.e., embodiments of the present invention such as embodiment of process1100. Comparing FIG. 15 to FIG. 16 shows that harvest numbers are morepredictable (as shown with FIG. 16) when rates are associated with cellpopulations that are expanding uniformly (i.e. well distributed duringseeding).

FIGS. 17 and 18 illustrate data for cells loaded and distributedconsistent with methods described above, i.e., embodiments of thepresent invention such as process 1100. FIG. 17 illustrates lactategeneration and FIG. 18 illustrates glucose consumption. FIGS. 17 and 18illustrate high run-to-run consistency. FIGS. 17 and 18 show doublingtime: Flask CV %=9.4, Embodiment of Present Invention CV %=2.4; andcells/cm2 at harvest: Flask CV %=28.0, Embodiment of Present InventionCV %=10.2. Cell population exponential growth is a sign of even celldistribution; particularly after 7 days of expansion.

FIGS. 19 and 20 illustrate data for cells loaded and distributed usingprevious methods. FIGS. 19 and 20 are provided for comparison. The factthat the best fit of the data in FIGS. 19 and 20 using regressionanalysis is the “power” regression line, is an indicator of a “constant”increase in lactate concentration. In contrast, there are exponentialincreases for methods consistent with embodiments of the presentinvention, as is illustrated in FIGS. 17 and 18.

Finally, FIG. 21 illustrates example components of a basic computersystem 1200 upon which embodiments of the present invention may beimplemented. Computer system 1200 may perform some steps in the methodsfor loading and distributing cells. System 1200 may be a controller forcontrolling features, e.g., flow control devices, pumps, valves,rotation of bioreactors, etc., of CES systems 10, 800, and 900 shownabove in which cells are loaded and distributed for expansion.

Computer system 1200 includes output device(s) 1204, and/or inputdevice(s) 1208. Output device(s) 1204 may include one or more displays,including CRT, LCD, and/or plasma displays. Output device(s) 1204 mayalso include a printer, speaker, etc. Input device(s) 1208 may include akeyboard, touch input devices, a mouse, voice input device, etc.

Basic computer system 1200 may also include a processing unit 1212and/or a memory 1216, according to embodiments of the present invention.The processing unit 1212 may be a general purpose processor operable toexecute instructions stored in memory 1216. Processing unit 1212 mayinclude a single processor or multiple processors, according toembodiments. Further, in embodiments, each processor may be a multi-coreprocessor having one or more cores to read and execute separateinstructions. The processors may include general purpose processors,application specific integrated circuits (ASICs), field programmablegate arrays (FPGAs), other integrated circuits.

The memory 1216 may include any tangible medium for short-term orlong-term storage for data and/or processor executable instructions,according to embodiments. The memory 1216 may include, for example,Random Access Memory (RAM), Read-Only Memory (ROM), or ElectricallyErasable Programmable Read-Only Memory (EEPROM). Other storage media mayinclude, for example, CD-ROM, tape, digital versatile disks (DVD) orother optical storage, tape, magnetic disk storage, magnetic tape, othermagnetic storage devices, etc. In embodiments, system 1200 may be usedto control the rotation of bioreactor 300 and/or various flow controldevices, pumps, valves, etc. of CES systems. Memory 1216 can storeprotocols 1220 and procedures 1224, such as protocols and procedures forloading and distributing cells in a bioreactor, which would controloperation of circulation pumps, valves, rotation of bioreactors, etc.

Storage 1228 may be any long-term data storage device or component.Storage 1220 may include one or more of the systems described inconjunction with memory 1216, according to embodiments. Storage 1228 maybe permanent or removable. In embodiments, system 1200 is part of a CESsystem and storage 1228 may store various protocols for utilizing theCES systems.

Various components may be referred to herein as “operably associated.”As used herein, “operably associated” refers to components that arelinked together in operable fashion, and encompasses embodiments inwhich components are linked directly, as well as embodiments in whichadditional components are placed between the two linked components.

The one or more present inventions may be embodied in other specificforms without departing from its spirit or essential characteristics.The described embodiments are to be considered in all respects only asillustrative and not restrictive. The scope of the invention is,therefore, indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope.

The one or more present inventions, in various embodiments, includecomponents, methods, processes, systems and/or apparatus substantiallyas depicted and described herein, including various embodiments,subcombinations, and subsets thereof. Those of skill in the art willunderstand how to make and use the present invention after understandingthe present disclosure.

The one or more present inventions, in various embodiments, includeproviding devices and processes in the absence of items not depictedand/or described herein or in various embodiments hereof, including inthe absence of such items as may have been used in previous devices orprocesses (e.g., for improving performance, achieving ease and/orreducing cost of implementation).

The foregoing discussion of the one or more present inventions has beenpresented for purposes of illustration and description. The foregoing isnot intended to limit the one or more present inventions to the form orforms disclosed herein. In the foregoing Detailed Description forexample, various features of the one or more present inventions aregrouped together in one or more embodiments for the purpose ofstreamlining the disclosure. This method of disclosure is not to beinterpreted as reflecting an intention that the claimed inventionrequires more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description, with eachclaim standing on its own as a separate preferred embodiment of the oneor more present inventions.

Moreover, though the description of the one or more present inventionshas included description of one or more embodiments and certainvariations and modifications, other variations and modifications arewithin the scope of the invention (e.g., as may be within the skill andknowledge of those in the art, after understanding the presentdisclosure). It is intended to obtain rights which include alternativeembodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1-39. (canceled)
 40. A system for expanding a plurality of cells, thesystem comprising: a bioreactor comprising a first fluid flow pathhaving at least opposing ends, a first opposing end of said first fluidflow path fluidly associated with a first port of a hollow fibermembrane and a second end of said first fluid flow path fluidlyassociated with a second port of the hollow fiber membrane, wherein saidfirst fluid flow path comprises an intracapillary portion of said hollowfiber membrane; a fluid inlet path fluidly associated with said firstfluid flow path, wherein the plurality of cells are introduced into thefirst fluid flow path through the first fluid inlet path; a first pumpfor circulating fluid in the first fluid flow path of the bioreactor;and a controller for controlling operation of the first pump, whereinthe controller is configured to control the pump to: circulate a fluidat a first rate within the first fluid flow path; maintain thecirculation rate of the fluid at the first rate for a predeterminedperiod of time to obtain a desired cell concentration in the fluid;after the desired cell concentration has been achieved, reduce thecirculation rate of the fluid to a reduced rate that is less than thefirst rate to allow a first percentage of the plurality of cells tosettle in the bioreactor and a second percentage of the plurality ofcells to settle outside of the bioreactor; circulate a fluid with growthmedia for expanding the first percentage of cells in the bioreactor andwasting most of the second percentage of cells from the first fluid flowpath.
 41. The system of claim 40, wherein the first percentage is equalto or greater than about 50 percent of the plurality of cells. 42-49.(canceled)
 50. The system of claim 40, wherein the first percentage isless than or equal to about 80 percent of the plurality of cells. 51.The system of claim 40, wherein the first rate is greater than or equalto about 25 ml/min. 52-61. (canceled)
 62. The system of claim 40,wherein the first rate is less than or equal to about 400 ml/min. 63-71.(canceled)